Method for purifying bioactive substances
a bioactive substance and purification method technology, applied in the field of purification methods of bioactive substances, can solve the problems of inability to stably immobilize his-tag proteins, difficult to perform quantitative purification at high purity levels using this purification technique, and inapplicability of techniques, etc., to achieve simple quantitative recovery of bioactive substances, high purity levels, and stable bonding
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Benefits of technology
Problems solved by technology
Method used
Image
Examples
example 1
(1) Active Esterification of CMD
[0088]CMD (by Meito Sangyo Co. Ltd., molecular weight: 1,000,000) was dissolved in ultrapure water such that the concentration thereof was 0.5 weight %. Then, a 0.4M EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) and 0.1M NHS (N-hydroxysuccinimide) solution was added to the CMD solution and stirred for five minutes at room temperature. The amounts of EDC and NHS are calculated to cause 2% of carboxyl groups to be activated in the case that the entire amount thereof undergoes a reaction.
(2) Immobilization of CMD onto Substrate
[0089]The activated esterified CMD solution was caused to react with EAH Sepharose 4B (by Pharmacia Biotech, Inc, 7-11 μmol / ml amino group concentration in swollen gel) using the method described in Affinity Chromatography Principles and Methods (Pharmacia Biotech) to immobilize the CMD onto the substrate.
(3) Formation of AB-NTA Film
[0090]The carboxyl groups of the CMD were activated, by adding 50 μl of a solution, which was ...
example 2
[0093]Preparation of the media and purification of His6-GFP were performed in the same manner as in Example 1, except that 1 mmol of EDC and 0.2 mmol of NHS were used to activate the carboxyl groups instead of 0.2 mmol of EDC and 0.04 mmol of NHS, and that 300 ml of a 10 mmol / L imidazole solution was used to wash the media instead of 500 ml of the 0.1 mmol / L imidazole solution.
example 3
[0094]Preparation of the media and purification of His6-GFP were performed in the same manner as in Example 1, except that a 1 nmol / L ferrous (II) chloride solution was used instead of the 1 mmol / L copper sulfate solution, and that 500 ml of a 1 mmol / L β-mercaptoethanol solution was used to wash the media instead of 500 ml of the 0.1 mmol / L imidazole solution.
PUM
| Property | Measurement | Unit |
|---|---|---|
| carbon number | aaaaa | aaaaa |
| thickness | aaaaa | aaaaa |
| thickness | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
Login to View More